Production of butanediol by anaerobic microbial fermentation

ABSTRACT

The invention provides methods for producing 2,3-butanediol by anaerobic fermentation. According to particular methods of the invention, 2,3-butanediol is produced by anaerobic fermentation of substrates including carbohydrate and carbon monoxide.

FIELD

The present invention relates to the production of butanediol bymicrobial fermentation, particularly to production of 2,3-butanediol bymicrobial fermentation of substrates comprising CO.

BACKGROUND

Biofuels for transportation are attractive replacements for gasoline andare rapidly penetrating fuel markets as low concentration blends.Biofuels, derived from natural plant sources, are more environmentallysustainable than those derived from fossil resources (such as gasoline),their use allowing a reduction in the levels of so-called fossil carbondioxide (CO₂) gas that is released into the atmosphere as a result offuel combustion. In addition, biofuels can be produced locally in manygeographies, and can act to reduce dependence on imported fossil energyresources. Alcohols suitable for use as biofuels include ethanol,butanol and 2,3-butanediol.

Ethanol is rapidly becoming a major hydrogen-rich liquid transport fuelaround the world. Worldwide consumption of ethanol in 2002 was anestimated 10.8 billion gallons. The global market for the fuel ethanolindustry is also predicted to grow sharply in future, due to anincreased interest in ethanol in Europe, Japan, the USA and severaldeveloping nations.

Butanediols including 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and2,3-butanediol may be considered to have a variety of advantages overethanol. Like ethanol, butanediols may be used directly as an automotivefuel additive. They may also be relatively easily transformed into anumber of other potentially higher value and/or higher energy products.For example, 2,3-butanediol may be readily converted in a two stepprocess into an eight-carbon dimer which can be used as aviation fuel.

2,3-Butanediol derives its versatility from its di-functional backbone,i.e., 2 hydroxyl groups are located at vicinal C-atoms allowing themolecule to be transformed quite easily into substances such asbutadiene, butadione, acetoin, methylethyl ketone etc. These chemicalcompounds are used as base molecules to manufacture a vast range ofindustrially produced chemicals.

In addition, 2,3-butanediol may be used as a fuel in an internalcombustion engine. It is in several ways more similar to gasoline thanit is to ethanol. As the interest in the production and application ofenvironmentally sustainable fuels has strengthened, interest inbiological processes to produce 2,3-butanediol (often referred to asbio-butanol) has increased.

2,3-Butanediol can be produced by microbial fermentation of carbohydratecontaining feedstock (Syu M J, Appl Microbial Biotechnol 55:10-18(2001), Qin et al., Chinese J Chem Eng 14(1):132-136 (2006)).2,3-Butanediol may also be produced by microbial fermentation of biomassfrom crops such as sugar beet, corn, wheat and sugarcane. However, thecost of these carbohydrate feed stocks is influenced by their value ashuman food or animal feed and the cultivation of starch orsucrose-producing crops for 2,3-butanediol production is noteconomically sustainable in all geographies. Therefore, it is ofinterest to develop technologies to convert lower cost and/or moreabundant carbon resources into 2,3-butanediol.

Carbon Monoxide (CO) is a major by-product of the incomplete combustionof organic materials such as coal or oil and oil derived products.Although the complete combustion of carbon containing precursors yieldsCO2 and water as the only end products, some industrial processes needelevated temperatures favouring the build up of carbon monoxide overCO2. One example is the steel industry, where high temperatures areneeded to generate desired steel qualities. For example, the steelindustry in Australia is reported to produce and release into theatmosphere over 500,000 tonnes of CO annually.

Furthermore, CO is also a major component of syngas, where varyingamounts of CO and H2 are generated by gasification of acarbon-containing fuel. For example, syngas may be produced by crackingthe organic biomass of waste woods and timber to generate precursors forthe production of fuels and more complex chemicals.

The release of CO into the atmosphere may have significant environmentalimpact. In addition, emissions taxes may be required to be paid,increasing costs to industrial plants. Since CO is a reactive energyrich molecule, it can be used as a precursor compound for the productionof a variety of chemicals. However, this valuable feedstock has not beenutilised to produce 2,3-butanediol.

It is an object of the present invention to provide a process that goesat least some way towards overcoming the above disadvantages or at leastto provide the public with a useful choice.

STATEMENT OF INVENTION

In one aspect, the invention provides a method of producing butanediolby microbial fermentation of a substrate comprising carbon monoxide. Inparticular embodiments, the invention provides a method of producingbutanediol by microbial fermentation, the method including:

-   -   a. providing a substrate comprising CO;    -   b. in a bioreactor containing a culture of one or more        micro-organisms, anaerobically fermenting the substrate to        produce butanediol.

In certain embodiments, the butanediol is 2,3-butanediol.

In another aspect, the invention provides a method of increasingefficiency of 2,3-butanediol production by fermentation, the methodincluding:

-   -   a. providing a substrate comprising CO;    -   b. in a bioreactor containing a culture of one or more        micro-organisms, anaerobically fermenting the substrate to        produce 2,3-butanediol.

In another aspect of the invention, there is provided a method ofproducing 2,3-butanediol by microbial fermentation, the methodincluding:

-   -   a. providing a substrate    -   b. in a bioreactor containing a culture of one or more        micro-organisms, anaerobically fermenting the substrate, wherein        one or more micro-organisms includes one or more 2,3-butanediol        dehydrogenase genes;    -   c. upregulating the 2,3-butanediol dehydrogenase gene(s), such        that 2,3-butanediol is produced by the micro-organism(s).

In particular embodiments, the substrate comprises CO.

In particular embodiments of the various aspects, the substratecomprising carbon monoxide is a gaseous substrate comprising carbonmonoxide. The gaseous substrate comprising carbon monoxide can beobtained as a by-product of an industrial process. In certainembodiments, the industrial process is selected from the groupconsisting of ferrous metal products manufacturing, non-ferrous productsmanufacturing, petroleum refining processes, gasification of biomass,gasification of coal, electric power production, carbon blackproduction, ammonia production, methanol production and cokemanufacturing. In one embodiment the gaseous substrate comprises a gasobtained from a steel mill. In another embodiment the gaseous substratecomprises automobile exhaust fumes.

In particular embodiments, the CO-containing substrate typicallycontains a major proportion of CO, such as at least about 20% to about100% CO by volume, from 40% to 95% CO by volume, from 40% to 60% CO byvolume, and from 45% to 55% CO by volume. In particular embodiments, thesubstrate comprises about 25%, or about 30%, or about 35%, or about 40%,or about 45%, or about 50% CO, or about 55% CO, or about 60% CO byvolume. Substrates having lower concentrations of CO, such as 6%, mayalso be appropriate, particularly when H₂ and CO₂ are also present.

In particular embodiments of the various aspects, the substratecomprising CO is provided at a sufficient level, such that2,3-butanediol is produced. In particular embodiments, CO is providedsuch that a specific uptake rate of at least 0.4 mmol/g/min; or at least0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min;or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min ismaintained.

In certain embodiments of the various aspects, the method comprisesmicrobial fermentation using Clostridium autoethanogenum.

In another aspect, the invention provides a method of producing2,3-butanediol by microbial fermentation, the method including:

-   -   a. providing a substrate    -   b. in a bioreactor including a culture of Clostridium        autoethanogenum, anaerobically fermenting the substrate to        produce 2,3-butanediol.

In particular embodiments, the substrate is one or more carbohydratessuch as fructose.

Alternatively the substrate is a substrate comprising carbon monoxide,typically a gaseous substrate comprising carbon monoxide, as hereinbefore described

In a further aspect, the invention provides a method for producingbutanediol by microbial fermentation of a first substrate and a secondsubstrate comprising CO. Preferably, the butanediol is 2,3-butanediol.

In particular embodiments, the first substrate is a carbohydrate. Incertain embodiments, the first substrate is fructose. In certainembodiments, the second substrate is a gaseous substrate comprisingcarbon monoxide, as herein before described.

In particular embodiments, the method includes the steps of:

-   -   (a) microbial fermentation of the first substrate to produce        2,3-butanediol    -   (b) microbial fermentation of the second substrate comprising CO        to produce 2,3-butanediol.

In certain embodiments, steps (a) and (b) may be conducted at the sametime. Alternatively, step (a) may substantially precede or follow step(b). In particular embodiments, the method may alternate between step(a) and step (b).

In a further aspect of the invention, there is provided a methodaccording to any of the previous aspects, wherein the fermentation iscarried out in a bioreactor.

In a further aspect of the invention, there is provided a methodaccording to any of the previous aspects, wherein the method furtherincludes the step of capturing or recovering the butanediol.

In a further aspect, there is provided butanediol, preferably2,3-butanediol, produced by the methods of any of the previous aspects.

The invention may also be said broadly to consist in the parts, elementsand features referred to or indicated in the specification of theapplication, individually or collectively, in any or all combinations oftwo or more of said parts, elements or features, and where specificintegers are mentioned herein which have known equivalents in the art towhich the invention relates, such known equivalents are deemed to beincorporated herein as if individually set forth.

DETAILED DESCRIPTION OF THE INVENTION

The following is a description of the present invention, includingpreferred embodiments thereof, given in general terms. The invention isfurther exemplified in the disclosure given under the heading “Examples”herein below, which provides experimental data supporting the invention,specific examples of aspects of the invention, and means of performingthe invention.

As used herein “butanediol” refers to all structural isomers of the diolincluding 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and2,3-butanediol and stereoisomers thereof. The term “2,3-butanediol”should be interpreted to include all enantiomeric and diastereomericforms of the compound, including (R,R), (S,S) and meso forms, inracemic, partially stereoisomerically pure and/or substantiallystereoisomerically pure forms.

The term “bioreactor” includes a fermentation device consisting of oneor more vessels and/or towers or piping arrangement, which includes theContinuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR),Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, StaticMixer, or other vessel or other device suitable for gas-liquid contact.As is described herein after, in some embodiments the bioreactor maycomprise a first growth reactor and a second fermentation reactor. Assuch, when referring to the addition of a substrate, for example asubstrate comprising carbon monoxide, to the bioreactor or fermentationreaction it should be understood to include addition to either or bothof these reactors where appropriate.

The term “substrate comprising carbon monoxide” and like terms should beunderstood to include any substrate in which carbon monoxide isavailable to one or more strains of bacteria for growth and/orfermentation, for example.

“Gaseous substrates comprising carbon monoxide” include any gas whichcontains a level of carbon monoxide. The gaseous substrate willtypically contain a major proportion of CO, preferably at least about15% to about 95% CO by volume.

Unless the context requires otherwise, the phrases “fermenting”,“fermentation process” or “fermentation reaction” and the like, as usedherein, are intended to encompass both the growth phase and productbiosynthesis phase of the process.

The inventors have surprisingly shown that 2,3-butanediol can beproduced by microbial fermentation using Clostridium autoethanogenum.They have found that fermentation products include a variety ofalcohols, whereby ethanol and 2,3-butanediol are significantsubstituents. 2,3-Butanediol has not been previously identified as afermentation product using Clostridium autoethanogenum. In particular,the inventors have determined that Clostridium autoethanogenum can beused to produce 2,3-butanediol and other products from a substratecomprising carbohydrate. In particular, fructose can be converted intoproducts including acetate, ethanol and 2,3-butanediol. It has also beensurprisingly demonstrated that 2,3-butanediol can be produced byClostridium autoethanogenum from substrates comprising CO, particularlygaseous substrates comprising CO. The use of a gaseous carbon source,particularly a source including CO, in fermentation processes has notpreviously resulted in the production of 2,3-butanediol.

In particular embodiments of the invention, the efficiency of2,3-butanediol production can be increased by providing the substrate ata sufficient level such that 2,3-butanediol is produced. It has beenrecognised that increasing the amount of substrate provided to amicrobial culture, increases the amount of 2,3-butanediol produced bythe culture.

In particular embodiments of the invention, the substrate comprising COis provided at a sufficient level such that 2,3-butanediol is produced.It has been shown that a microbial culture comprising C. autoethanogenumcan uptake CO at a rate up to approximately 1.5 to 2 mmol/gram dryweight microbial cells/minute (specific CO uptake). In particularembodiments of the invention, a substrate comprising CO is provided tothe microbial culture comprising C. autoethanogenum such that a specificuptake is maintained substantially at or at least 0.4 mmol/g/min; or atleast 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; orat least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5mmol/g/min. In such embodiments, 2,3-butanediol is a significantfermentation product of at least 0.5 g/L; or at least 1 g/L; or at least2 g/L; or at least 5 g/L. In particular embodiments, 2,3-butanediol isproduced at a rate of at least 0.5 g/L/day; or at least 1 g/L/day.

In particular embodiments of the invention, apparatus used forconducting methods of the invention enable measurement and/or control ofparameters such as CO supply, CO uptake, biomass level, 2,3-butanediolproduction. For example, samples can be taken from a bioreactor todetermine one or more of the above parameters and the bioreactorconditions optionally adjusted to improve 2,3-butanediol production. Forexample, in a bioreactor, wherein the microbial culture is producing noor insignificant amounts of 2,3-butanediol, the CO supply can beincreased such that 2,3-butanediol is produced.

It is accepted that products such as acetate and ethanol are producedfrom CO via a combination of the acetyl-CoA cycle and the THF cycle asdescribed in Phillips, J. R, et al, 1994, Applied Biochemistry andBiotechnology, 45/46: 145. However, in accordance with the methods ofthe invention, it has been surprisingly shown that 2,3-butanediol can beproduced, particularly where CO is provided such that specific CO uptakerates of at least 0.4 mmol/g/min; or at least 0.5 mmol/g/min; or atleast 0.6 mmol/g/min; or at least 0.7 mmol/g/min; or at least 0.8mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0 mmol/g/min; orat least 1.2 mmol/g/min; or at least 1.5 mmol/g/min are maintained.Without wishing to be bound by theory, it is considered that byproviding sufficient or elevated levels of CO, higher energy products,such as 2,3-butanediol can be produced during fermentation. It isconsidered precursors of products, such as 2,3-butanediol act aselectron acceptors to relieve the microbial cell of excess reducingpower, in the form of NAD(P)H, thus restoring a favourableNAD(P):NAD(P)H equilibrium. It is further considered that carbohydratesfermented by the culture can also be converted into 2,3-butanediol in asimilar manner.

The following genes have been putatively identified in C.autoethanogenum: a-acetolactate synthase (ALS), α-acetolactatedecarboxylase (ALDC) and 2,3-butanediol dehydrogenase (2,3BDH). Theputative 2,3-butanediol dehydrogenase gene (ORF 1283) of C.autoethanogenum (strain deposited at DSMZ under the accession number19630) shows strong homology to the 2,3BDH of Clostridium novyi(NT01CX_(—)0344) with amino acid identities of 73% (262/357) andpositives of 84% (300/357). ORF 1283 also shows significant homology tothe gene YdjL (bdhA) of Bacillus subtilis (47% amino acid identities,63% positives and E-value of 3e-89. Further evidence that ORF 1283 ofLZ1560 is 2,3BDH comes from homology to 2,3BDH(YAL060W) of Saccharomycescerevisiae (E=2e−53).

Without wishing to be bound by theory, it is considered 2,3-butanediolis produced from pyruvate (an intermediate in anabolism produced fromacetyl CoA) as follows:

Real-Time PCR studies of 2,3-butanediol dehydrogenase in C.autoethanogenum, indicate that it is substantially upregulated incultures where significant amounts of 2,3-butanediol are produced. Thus,2,3-butanediol dehydrogenase can be upregulated in accordance with themethods of the invention. For example, where CO is supplied atsufficient levels, 2,3-butanediol dehydrogenase is upregulated. Inparticular, where CO is supplied such that the specific CO uptake by themicrobial culture is at least 0.4 mmol/g/min; or at least 0.5mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7 mmol/g/min; orat least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; or at least 1.0mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5 mmol/g/min;2,3-butanediol dehydrogenase is upregulated. As such, the inventionprovides a method of producing 2,3-butanediol by microbial fermentationof a substrate by upregulation of 2,3-butanediol dehydrogenase.

The inventors have further demonstrated that different substrates, suchas a carbohydrate substrate and a gaseous substrate comprising CO, canbe switched during microbial production of 2,3-butanediol, withoutdeleterious effect. In addition, they contemplate that substrates couldbe alternated, for example when one substrate is unavailable, and wouldcontinue to produce 2,3-butanediol.

In accordance with the results obtained, in one embodiment of theinvention, 2,3-butanediol is produced by microbial fermentation of asubstrate comprising carbohydrate. In another embodiment of theinvention, a substrate comprising carbon monoxide, preferably a gaseoussubstrate comprising CO, is converted into various products including2,3-butanediol, by Clostridium autoethanogenum.

In a further embodiment of the invention, a first substrate comprisingcarbohydrate (preferably fructose) may be used in initial stages of thefermentation reaction and following complete consumption of thesubstrate, the substrate can be switched to a second substratecomprising CO. Again, the inventors have surprisingly determined that2,3-butanediol is produced in the initial stages where the firstsubstrate comprising carbohydrate is the sole carbon source and is alsoproduced in the latter stages where the substrate comprising CO is thesole carbon source.

The inventors have shown that 2,3-butanediol is produced under a varietyof conditions, including media containing alternative buffer solutionssuch as acetate buffer and citrate buffer. The inventors also submitthat in embodiments where the pH is uncontrolled and may be variable,2,3-butanediol is still produced. Examples of media suitable forcarrying out the desired fermentation are described in the examplessection hereinafter.

The inventors contemplate that the 2,3-butanediol produced in suchprocesses may be readily recovered using separation techniques known inthe art. Furthermore, the 2,3-butanediol may be readily converted intosubstances such as butadiene, butadione, acetoin, methylethyl ketone andthe like. Such chemical compounds are valuable base molecules used tomanufacture a significant percentage of all chemical industry products.Therefore, the inventors contemplate that the 2,3-butanediol produced inthe processes disclosed herein may be used in the manufacture of a widerange of well known industrial products.

The invention is generally described herein in relation to preferredembodiments of the invention which utilise Clostridium autoethanogenumand/or produce 2,3-butanediol. However, it should be appreciated thatalternative micro-organisms may be substituted for C. autoethanogenum.Similarly, the methods may be used to produce and recover butanediolsother than 2,3-butanediol. Accordingly, unless the context requiresotherwise, reference to “2,3-butanediol” may be substituted by thegeneral term “butanediol”.

Method

In one embodiment, the invention provides a method for the production ofbutanediol by microbial fermentation. In a preferred embodiment themethod comprises at least the step of anaerobically fermenting asubstrate comprising CO, preferably a gaseous substrate comprising CO,to obtain 2,3-butanediol.

In a particular embodiment of the invention, the method includes thesteps of:

-   -   (a) providing a substrate comprising CO, preferably a gaseous        substrate comprising CO;    -   (b) in a bioreactor containing a culture of one or more        micro-organisms anaerobically fermenting the substrate to        produce 2,3-butanediol.

In another embodiment, the invention provides a method of increasingefficiency of 2,3-butanediol production by fermentation, the methodincluding:

-   -   (a) providing a substrate comprising CO;    -   (b) in a bioreactor containing a culture of one or more        micro-organisms, anaerobically fermenting the substrate to        produce 2,3-butanediol.

In particular embodiments, the substrate comprising CO is provided at alevel sufficient to produce significant amounts of 2,3-butanediol, suchas at least 0.5 g/L of fermentation media, or at least 1 g/L, or atleast 2 g/L, or at least 5 g/L. In certain embodiments, CO is providedat a level sufficient to produce 2,3-butanediol at a rate of at least0.5 g/L/day; or at least 1 g/L/day. In particular embodiments, CO isprovided such that a specific uptake rate of at least 0.4 mmol/g/min; orat least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; orat least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5mmol/g/min is maintained. Those skilled in the art will appreciatemethods of supplying CO, particularly gaseous CO, such that the requireduptake rate is achieved. However, by way of example, factors such asincreasing gas hold-up in a fermentation media will increase the amountof CO available for conversion to products by the microbial culture. Gashold-up can typically be increased by mechanical means, such asincreasing agitation in a CSTR. Furthermore, supplying CO at a fasterrate or a higher partial pressure will also increase the CO availabilityin a fermentation broth.

In another embodiment, the method involves fermentation of a substratecomprising carbohydrate by Clostridium autoethanogenum to producebutanediol, preferably, 2,3-butanediol.

In another embodiment, the method includes the steps of:

-   -   (a) microbial fermentation of the first substrate to produce        2,3-butanediol    -   (b) microbial fermentation of the second substrate comprising CO        to produce 2,3-butanediol.

In certain embodiments, the first substrate is carbohydrate and in someembodiments, the substrate is fructose. Preferably, the second substrateis a gaseous substrate comprising CO. In particular embodiments, steps(a) and (b) may be conducted at the same time. Alternatively, step (a)may substantially precede or follow step (b). Preferably, the method mayalternate between step (a) and step (b).

In certain embodiments of the invention, the method further includes thestep of capturing or recovering the 2,3-butanediol produced.

Micro-Organisms

In embodiments of the invention the one or more micro-organisms used inthe fermentation is Clostridium autoethanogenum. In a preferredembodiment the Clostridium autoethanogenum is a Clostridiumautoethanogenum having the identifying characteristics of the straindeposited at the German Resource Centre for Biological Material (DSMZ)under the identifying deposit number 19630. In another embodiment theClostridium autoethanogenum is a Clostridium autoethanogenum having theidentifying characteristics of DSMZ deposit number DSMZ 10061.

Culturing of the bacteria used in a method of the invention may beconducted using any number of processes known in the art for culturingand fermenting substrates using anaerobic bacteria. Exemplary techniquesare provided in the “Examples” section of this document. By way offurther example, those processes generally described in the followingarticles using gaseous substrates for fermentation may be utilised: K.T. Klasson, M. D. Ackerson, E. C. Clausen and J. L. Gaddy (1991).Bioreactors for synthesis gas fermentations resources. Conservation andRecycling, 5; 145-165; K. T. Klasson, M. D. Ackerson, E. C. Clausen andJ. L. Gaddy (1991). Bioreactor design for synthesis gas fermentations.Fuel. 70. 605-614; K. T. Klasson, M. D. Ackerson, E. C. Clausen and J.L. Gaddy (1992). Bioconversion of synthesis gas into liquid or gaseousfuels. Enzyme and Microbial Technology. 14; 602-608; J. L. Vega, G. M.Antorrena, E. C. Clausen and J. L. Gaddy (1989). Study of GaseousSubstrate Fermentation: Carbon Monoxide Conversion to Acetate. 2.Continuous Culture. Biotech. Bioeng. 34. 6. 785-793; J. L. Vega, E. C.Clausen and J. L. Gaddy (1989). Study of gaseous substratefermentations: Carbon monoxide conversion to acetate. 1. Batch culture.Biotechnology and Bioengineering. 34. 6. 774-784; and, J. L. Vega, E. C.Clausen and J. L. Gaddy (1990). Design of Bioreactors for Coal SynthesisGas Fermentations. Resources, Conservation and Recycling. 3. 149-160.Methods for culturing bacteria on substrates comprising carbohydratesare also well known in the art.

Substrates

In one embodiment of the invention, 2,3-butanediol is produced bymicrobial fermentation of a substrate comprising carbohydrate usingClostridium autoethanogenum. It will be appreciated there are manyexamples of carbohydrates suitable for fermentation known in the art andmany examples of the types of processes used to ferment the carbohydratesubstrate. By way of example, suitable substrates may include, but arenot limited to, monosaccharides such as glucose and fructose,oligosaccharides such as sucrose or lactose, polysaccharides, such ascellulose or starch. Although it is contemplated that any of thesecarbohydrate substrates (and mixtures thereof) are suitable in thepresent invention, preferred carbohydrate substrates are fructose andsucrose (and mixtures thereof).

Those skilled in the art will appreciate fermentable sugars may beobtained from cellulosic and lignocellulosic biomass through processesof pre-treatment and saccharification, as described, for example, inUS20070031918. Biomass refers to any cellulose or lignocellulosicmaterial and includes materials comprising cellulose, and optionallyfurther comprising hemicellulose, lignin, starch, oligosaccharidesand/or monosaccharides. Biomass includes, but is not limited tobioenergy crops, agricultural residues, municipal solid waste,industrial solid waste, sludge from paper manufacture, yard waste, woodand forestry waste. However, in exemplary embodiments of the inventioncommercially available fructose is used as the carbon and energy sourcefor the fermentation.

In a particular embodiment, a substrate comprising carbon monoxide,preferably a gaseous substrate comprising carbon monoxide is used in themethods of the invention. The gaseous substrate may be a waste gasobtained as a by-product of an industrial process, or from some othersource such as from combustion engine (for example automobile) exhaustfumes. In certain embodiments, the industrial process is selected fromthe group consisting of ferrous metal products manufacturing, such as asteel mill, non-ferrous products manufacturing, petroleum refiningprocesses, gasification of coal, electric power production, carbon blackproduction, ammonia production, methanol production and cokemanufacturing. In these embodiments, the CO-containing gas may becaptured from the industrial process before it is emitted into theatmosphere, using any convenient method. Depending on the composition ofthe gaseous substrate comprising carbon monoxide, it may also bedesirable to treat it to remove any undesired impurities, such as dustparticles before introducing it to the fermentation. For example, thegaseous substrate may be filtered or scrubbed using known methods.

In other embodiments of the invention, the gaseous substrate comprisingcarbon monoxide may be sourced from the gasification of biomass. Theprocess of gasification involves partial combustion of biomass in arestricted supply of air or oxygen. The resultant gas typicallycomprises mainly CO and H₂, with minimal volumes of CO₂, methane,ethylene and ethane. For example, biomass by-products obtained duringthe extraction and processing of foodstuffs such as sugar fromsugarcane, or starch from maize or grains, or non-food biomass wastegenerated by the forestry industry may be gasified to produce aCO-containing gas suitable for use in the present invention.

The CO-containing substrate will typically contain a major proportion ofCO, such as at least about 20% to about 100% CO by volume, from 40% to95% CO by volume, from 40% to 60% CO by volume, and from 45% to 55% COby volume. In particular embodiments, the substrate comprises about 25%,or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO,or about 55% CO, or about 60% CO by volume. Substrates having lowerconcentrations of CO, such as 6%, may also be appropriate, particularlywhen H₂ and CO₂ are also present.

In particular embodiments, CO is supplied at a level sufficient for2,3-butanediol production to occur. In particular embodiments, CO isprovided such that a specific uptake rate of at least 0.4 mmol/g/min; orat least 0.5 mmol/g/min; or at least 0.6 mmol/g/min; or at least 0.7mmol/g/min; or at least 0.8 mmol/g/min; or at least 0.9 mmol/g/min; orat least 1.0 mmol/g/min; or at least 1.2 mmol/g/min; or at least 1.5mmol/g/min is maintained. Those skilled in the art will appreciatemethods of supplying CO, particularly gaseous CO, such that the requireduptake rate is achieved. However, by way of example, factors such asincreasing gas hold-up in a fermentation media will increase the amountof CO available for conversion to products by the microbial culture.Those skilled in the art will appreciate methods of increasing gashold-up. However, by way of non-limiting example, gas hold-up istypically increased by mechanical means such as increasing agitation ina CSTR. Furthermore, supplying CO at a faster rate or a higher partialpressure will also increase the CO availability in a fermentation broth.

It is not necessary for the gaseous substrate to contain any hydrogen,however this is not considered detrimental to 2,3-butanediol production.The gaseous substrate may also contain some CO₂ for example, such asabout 1% to about 80% by volume, or 1% to about 30% by volume. In oneembodiment it contains about 5% to about 10% by volume. In anotherembodiment the gaseous substrate contains approximately 20% CO₂ byvolume.

Typically, the carbon monoxide will be added to the fermentationreaction in a gaseous state. However, the invention should not beconsidered to be limited to addition of the substrate in this state. Forexample, the carbon monoxide could be provided in a liquid. For example,a liquid may be saturated with a carbon monoxide containing gas and thenthat liquid added to a bioreactor. This may be achieved using standardmethodology. By way of example a microbubble dispersion generator(Hensirisak et. al. Scale-up of microbubble dispersion generator foraerobic fermentation; Applied Biochemistry and Biotechnology Volume 101,Number 3/October, 2002) could be used.

In one embodiment of the invention, the inventors have determined that2,3-butanediol can be produced by fermentation of a first substrate anda second substrate. In one particular embodiment of the invention,2,3-butanediol will be produced when a first substrate, for example acarbohydrate such as fructose and a second substrate, preferably asubstrate comprising CO, are provided.

In a further embodiment, the inventors have determined that2,3-butanediol will be produced by a first substrate and on completeconsumption, the first substrate may be replaced with a second substrateand the 2,3-butanediol continues to be produced. In a particularembodiment, the first substrate is fructose and on complete consumptionof the fructose, a substrate comprising CO can be provided. Theinventors have surprisingly found that 2,3-butanediol continues to beproduced. The inventors further contemplate that the first substrate andsecond substrate may be alternated if needed. For example if a firstsubstrate is unavailable, an alternative substrate may be used until theavailability of the first substrate improves.

Media

It will be appreciated that for growth of the bacteria and substrate tobutanediol fermentation to occur, in addition to the substrate, asuitable nutrient medium will need to be fed to the bioreactor. Anutrient medium will contain components, such as vitamins and minerals,sufficient to permit growth of the micro-organism used. Anaerobic mediasuitable for the growth of Clostridium autoethanogenum are known in theart, as described for example by Abrini et al (Clostridiumautoethanogenum, sp. Nov., An Anaerobic Bacterium That Produces EthanolFrom Carbon Monoxide; Arch. Microbiol., 161: 345-351 (1994)). The“Examples” section herein after provides further examples of suitablemedia.

Fermentation Conditions

The fermentation should desirably be carried out under appropriateconditions for the substrate to butanediol fermentation to occur.Reaction conditions that should be considered include temperature, mediaflow rate, pH, media redox potential, agitation rate (if using acontinuous stirred tank reactor), inoculum level, maximum substrateconcentrations and rates of introduction of the substrate to thebioreactor to ensure that substrate level does not become limiting, andmaximum product concentrations to avoid product inhibition. Examples offermentation conditions suitable for anaerobic fermentation of asubstrate comprising CO are detailed in WO2007/117157, WO2008/115080,WO2009/022925 and WO2009/064200, the disclosure of which areincorporated herein by reference. It is recognised the fermentationconditions reported therein can be readily modified in accordance withthe methods of the instant invention.

The inventors have determined that, in one embodiment where pH is notcontrolled, there does not appear to be a deleterious effect on2,3-butanediol production.

Bioreactor

Fermentation reactions may be carried out in any suitable bioreactor asdescribed previously herein. In some embodiments of the invention, thebioreactor may comprise a first, growth reactor in which themicro-organisms are cultured, and a second, fermentation reactor, towhich broth from the growth reactor is fed and in which most of thefermentation product (2,3-butanediol, for example) is produced.

Product Recovery

The fermentation will result in a fermentation broth comprising adesirable product (such as butanediol) and/or one or more by-products(such as ethanol, acetate and butyrate) as well as bacterial cells, in anutrient medium. In a preferred embodiment, the fermentation productsinclude 2,3-butanediol.

2,3-butanediol, or a mixed alcohol stream containing 2,3-butanediol andone or more other alcohols, may be recovered from the fermentation brothby methods known in the art, such as fractional distillation orevaporation, pervaporation, and extractive fermentation. By-productssuch as acids including acetate and butyrate may also be recovered fromthe fermentation broth using methods known in the art. For example, anadsorption system involving an activated charcoal filter orelectrodialysis may be used.

In certain embodiments of the invention, 2,3-butanediol and by-productsare recovered from the fermentation broth by continuously removing aportion of the broth from the bioreactor, separating microbial cellsfrom the broth (conveniently by filtration, for example), and recovering2,3-butanediol and optionally other alcohols and acids from the broth.Alcohols may conveniently be recovered for example by distillation, andacids may be recovered for example by adsorption on activated charcoal.The separated microbial cells are preferably returned to thefermentation bioreactor. The cell free permeate remaining after thealcohol(s) and acid(s) have been removed is also preferably returned tothe fermentation bioreactor. Additional nutrients (such as B vitamins)may be added to the cell free permeate to replenish the nutrient mediumbefore it is returned to the bioreactor.

Also, if the pH of the broth was adjusted during recovery of2,3-butanediol and/or by-products, the pH should be re-adjusted to asimilar pH to that of the broth in the fermentation bioreactor, beforebeing returned to the bioreactor.

The invention will now be described in more detail with reference to thefollowing non-limiting examples.

EXAMPLES Materials and Methods

Solution A NH₄Ac 3.083 g KCl 0.15 g MgCl₂•6H₂O 0.61 g NaCl (optional)0.12 g CaCl₂•2H₂O 0.294 g Distilled Water Up to 1 L Solution B Biotin20.0 mg Calcium D-(*)- 50.0 mg pantothenate Folic acid 20.0 mg VitaminB12 50.0 mg Pyridoxine. HCl 10.0 mg p-Aminobenzoic acid 50.0 mgThiamine. HCl 50.0 mg Thioctic acid 50.0 mg Riboflavin 50.0 mg Distilledwater To 1 Litre Nicotinic acid 50.0 mg Solution(s) C Component/0.1MQuantity/ml Component/0.1M Quantity/ml solution (aq) into 1 L mediasolution (aq) into 1 L media FeCl₃  1 ml MnCl₂ 0.1 ml CoCl₂ 0.5 mlNa₂WO₄ 0.1 ml NiCl₂ 0.5 ml ZnCl₂ 0.1 ml H₃BO₃ 0.1 ml Na₂SeO₃ 0.1 mlNa₂MoO₄ 0.1 ml Solution D Solution E Concentration per Concentration perMedia Component 1.0 L of Media 1.0 L of Media MgCl₂•6H₂O 0.5 g 0.5 gNaCl 0.2 g 0.2 g CaCl₂ 0.2 g 0.2 g 100 mM sodium — 160 mL phosphatebuffer (pH 6.0) NaH₂PO₄ 2.04 g — NH₄Cl 2.5 g 0.6 g 85% H₃PO₄ — 0.5 mLKCl 0.15 g 0.15 g Solution C 10 mL 10 mL Solution E 10 mL 10 mLResazurin (1000 mg/L 2 mL 1 mL stock) FeCl₃ 0.01 g 0.0025 g Cysteine HCl0.5 g 0.25 g monohydrate Agarose (optional) 15 g — Distilled water To 1Litre To 1 Litre Solution F Composite trace metal solution per L ofstock Nitrilotriacetic Acid 1.5 g MgSO₄•7H₂O 3.0 g MnSO₄•H₂O 0.5 g NaCl1.0 g FeSO₄•7H₂O 0.1 g Fe(SO₄)₂(NH₄)₂•6H₂O 0.8 g CoCl₂•6H₂O 0.2 gZnSO₄•7H₂O 0.2 g CuCl₂•2H₂O 0.02 g AlK(SO₄)₂•12H₂O 0.02 g H₃BO₃ 0.30 gNaMoO₄•2H₂O 0.03 g Na₂SeO₃ 0.02 g NiCl₂•6H₂O 0.02 g Na₂WO₄•6H₂O 0.02 gDistilled water To 1 Litre

Preparation of Na₂S_(x)

A 500 ml flask was charged with Na₂S (93.7 g, 0.39 mol) and 200 ml H₂O.The solution was stirred until the salt had dissolved and sulfur (25 g,0.1 mol) was added under constant N₂ flow. After 2 hours stirring atroom temperature, the “Na₂S_(x)” solution (approx 4M with respect to[Na] and approx 5M with respect to sulfur), now a clear reddish brownliquid, was transferred into N₂ purged serum bottles, wrapped inaluminium foil.

Preparation of Cr (II) Solution

A 1 L three necked flask was fitted with a gas tight inlet and outlet toallow working under inert gas and subsequent transfer of the desiredproduct into a suitable storage flask. The flask was charged withCrCl₃.6H₂O (40 g, 0.15 mol), zinc granules [20 mesh] (18.3 g, 0.28 mol),mercury (13.55 g, 1 mL, 0.0676 mol) and 500 mL of distilled water.Following flushing with N₂ for one hour, the mixture was warmed to about80° C. to initiate the reaction. Following two hours of stirring under aconstant N₂ flow, the mixture was cooled to room temperature andcontinuously stirred for another 48 hours by which time the reactionmixture had turned to a deep blue solution. The solution was transferredinto N₂ purged serum bottles and stored in the fridge for future use.

Bacteria:

The Clostridium autoethanogenum used is that deposited at the GermanResource Centre for Biological Material (DSMZ) and allocated theaccession number 19630.

Sampling and Analytical Procedures

Media samples were taken from the fermentation reactor (e.g. CSTR orserum bottle) at intervals over the course of the fermentation. Eachtime the media was sampled care was taken to ensure that no gas wasallowed to enter into or escape from the reactor.

HPLC:

HPLC System Agilent 1100 Series. Mobile Phase: 0.0025N Sulfuric Acid.Flow and pressure: 0.800 mL/min. Column: Alltech IOA; Catalog #9648,150×6.5 mm, particle size 5 μm. Temperature of column: 60° C. Detector:Refractive Index. Temperature of detector: 45° C.

Method for Sample Preparation:

400 μL of sample and 50 μL of 0.15M ZnSO₄ and 50 μL of 0.15M Ba(OH)₂ areloaded into an Eppendorf tube. The tubes are centrifuged for 10 min. at12,000 rpm, 4° C. 200 μl of the supernatant are transferred into an HPLCvial, and 54 are injected into the HPLC instrument.

Headspace Analysis:

Measurements were carried out on a Varian CP-4900 micro GC with twoinstalled channels. Channel 1 was a 10 m Mol-sieve column running at 70°C., 200 kPa argon and a backflush time of 4.2 s, while channel 2 was a10 m PPQ column running at 90° C., 150 kPa helium and no backflush. Theinjector temperature for both channels was 70° C. Runtimes were set to120 s, but all peaks of interest would usually elute before 100 s.Specific CO uptake was determined by calculating CO consumption per gramof cells (dry weight—see below).

Cell Density:

Cell density was determined by counting bacterial cells in a definedaliquot of fermentation broth. Alternatively, the absorbance of thesamples was measured at 600 nm (spectrophotometer) and the dry weightdetermined via calculation according to published procedures.

Metal Sulfide Solution 1:

Approximately 950 mL of solution A was transferred into a 1 L fermenterand sparged with nitrogen. H₃PO₄ (85% solution, 1.5 mL) was added andthe pH adjusted to 5.3 using concentrated NH₄OH(aq). Resazurin (1 mL ofa 2 g/L solution) was added and the solution sparged with N2.Chromium(II)chloride was added until the ORP of the solution decreasedto approximately −150 mV. 10×Solution(s) C were added before sodiumpolysulfide (1.44 mL of a 4.3M or 1 mL of a 6M solution) were added andthe solution sparged with N2.

Example 1A Production of 2,3-butanediol by fermentation

Fermentive conversion of a substrate, using Clostridium autoethanogenumwas conducted in a CSTR reactor over a two week period, with periodicmonitoring. The media used for the CSTR experiments were prepared inaccordance with the components listed in Table E. The phosphate saltmixture consisted of 0.65 mM Na₂HPO4 and 15.3 mM NaH₂PO₄. All othercomponents such as the phosphoric acid, the ammonium salts and thecysteine-hydrochloride were mixed into 800 ml of water before the buffersalts were added to the solution. Proceeding in this manner ensured thatthe pH increased above about 6.5 avoiding the precipitation of mediacomponents. The solution was diluted to 1 L and made anaerobic byheating to boiling and allowing it to cool to room temperature under aconstant flow of N2 gas. Once cool, the solution was adjusted to thefinal pH of 5.3 and the B vitamins added. Anaerobicity was maintainedthroughout the experiment. Carbohydrate (5 g/L fructose) was added tothe basic media formulation. The media solutions were introduced intothe fermenters and optionally sparged with the respective CO containinggases from the start of the experiment, or after a predeterminedinterval. During these experiments, the pH was controlled to remain at5.5 by adding an aqueous solution of NaOH. An actively growingClostridium autoethanogenum culture was inoculated into the reactor at alevel of 5% (v/v). The temperature of the reactor was maintained at 37°C. and agitation rate was 400 rpm.

Results:

Initially, the fermentation contained fructose as a substrate, whichresulted in the production of acetic acid, ethanol and 2,3-butanediol.Over time, the fructose was consumed and a gas stream including CO (95%CO, 5% CO2) was sparged through the media. The media was maintained atpH 5.5 (Table 1). It should be noted that even when the carbohydrate hadbeen consumed, the above mentioned products increased in concentration,clearly demonstrating that the CO was used to produce the productsincluding 2,3-butanediol.

TABLE 1 Monitoring of production of 2,3-butanediol, ethanol and acetate(concentrations in g/L) over time in a CSTR reactor. Time\hours FructoseAcetic acid Ethanol 2,3-Butanediol 0 5 0 0 0 23 5 0.123 0.018 0 45 3.80.579 0.167 0.05 110 0 4.753 2.8 1.2 185 0 7.2 3.8 1.9 324 0 6.736 4.91.91

Example 1B Production of 2,3-butanediol by Fermentation

In a further experiment, the conversion of a substrate by Clostridiumautoethanogenum was conducted in a CSTR reactor over a 10 day period,with periodic monitoring. In this instance the fermenter and media wereprepared in accordance with Example 1A, however the substrate wasexclusively simulated steel mill gas (70% CO, 1% H2, 14% N2, 15% CO2)sparged continuously and the pH of the media was kept constant at 5.5(Table 2). Conversion of the substrate again resulted in acetic acid,ethanol and 2,3-butanediol, demonstrating that even in the absence of acarbohydrate substrate at the commencement of the fermentation, aceticacid, ethanol and butanediol are produced.

TABLE 2 Monitoring of production of 2,3-butanediol, ethanol and acetate(concentrations in g/L) over time in a CSTR batch reactor. Time\daysAcetic acid Ethanol 2,3-Butanediol 0 0 0 0 6 4.5 0.5 0 10 5 4 0.5

Example 2 Production of 2,3-butanediol by Fermentation

In a further experiment, the conversion of a substrate by Clostridiumautoethanogenum was conducted in a CSTR reactor over a 3 day period,with periodic monitoring. In this instance, the fermenter and media wereprepared in accordance with that described in Example 1A, however thesubstrate was simulated steel mill gas (70% CO, 1% H2, 14% N2, 15% CO2),sparged continuously and fructose (10 g/L) and the pH of the media waskept constant at 5.5 (Table 3). Conversion of the substrate againresulted in acetic acid, ethanol and 2,3-butanediol.

TABLE 3 Monitoring of production of 2,3-butanediol, ethanol and acetate(concentrations in g/L) over time in a CSTR batch reactor. Time\hoursFructose Acetic acid Ethanol 2,3-Butanediol 0 10 0 0 0 15 9.8 0.8 0.20.05 23 8.87 1.7 0.7 0.2 39 5.3 3.7 2.3 0.9 69 1.8 7.3 4 3.1

The final concentrations of acetate, ethanol and 2,3-butanediol werealso compared between the fermenter experiments outlined in Examples 1A,1B and 2, at the end of each experiment (note, these results relate tothe final concentrations measured in tables 1-3 and are summarised forcomparison in Table 4).

TABLE 4 Examples of 2,3-butanediol production using alternativesubstrates in a CSTR reactor, measured on conclusion of each experiment.Final Final Final 2,3- Fermentation Substrate Acetate Ethanol Butanedioltime (days) Fructose then Mill 6.7 4.9 1.9 13.5 gas (Ex 1A) Mill gasonly (Ex 1B) 5 4 0.5 10 Fructose and Mill 7.3 4 3.1 2.9 gas (Ex 2)Results are given in g/L.

Example 3 Production of 2,3-butanediol by Fermentation

In order to ascertain how media conditions may affect the production of2,3-butanediol, serum bottles containing media comprising a selection ofbuffers were prepared and the fermentation products analysed at the endof the experiment (Table 5). Incubation was performed in 234 ml sealedserum bottles each containing 50 ml of the above described media (TableE), optionally buffered with either an acetate buffer (0.02M) or acitrate buffer (0.02M) and adjusted to pH 5.3. The 184 ml headspace ofeach serum bottle was initially N2 and then filled to an overpressure of30 psi with either 95% CO, 5% CO2, or 70% CO, 15% CO2, 14% N2, 1% H2.Each bottle was inoculated with 2 ml of a Clostridium autoethanogenumculture. A shaking incubator was used and the reaction temperature wasmaintained at 37° C.

Once again, it is clear that 2,3-butanediol is produced irrespective ofthe buffer used in the experiment. Furthermore, it should also be notedthat since the serum bottles were not pH controlled, the product alsoappeared to be produced with limited (or no) control of pH.

TABLE 5 Examples of 2,3-butanediol production in a variety of media.Media used and Final Final Final 2,3- system Acetate Ethanol ButanediolSerum Bottle 0.02M 5.597 1.1 0.43 acetate buffer Serum Bottle 0.02M6.547 0.364 0.16 Citrate buffer The media of the serum bottles wereanalysed after the active growth, i.e., the increase in cell masslevelled out after several days (4 to 7 days). Results are given in g/L.

Example 4 Batch Fermentation in CSTR

Approximately 1.3 L of solution A was transferred into a 2 L fermenterand sparged with nitrogen. Resazurin (1.35 mL of a 2 g/L solution) wasadded. H₃PO₄ (85% solution, 2.025 mL) was added and the pH adjusted to5.3 using conc. NH₄OH(aq). Chromium(II)chloride was added until the ORPof the solution decreased to approximately −150 mV. Sodium polysulfide(6.07 ml of a 4.3M solution) was added and the pH adjusted to 5.5 usingconcentrated HCl. The solution was sparged with N2 for 1 hour before theaddition of metal sulfide solution 1 (150 ml) and Solution B (15 ml).The solution was sparged with N2 then CO containing gas (3% H2; 30% N2;47% CO; 20% CO2), before inoculation with an actively growingClostridium autoethanogenum culture at a level of approximately 5%(v/v). Gas flow rate was adjusted to ensure the microbial culture wasnot limited in CO in order to maintain a high specific CO uptake.Results of the fermentation are shown in Table 6.

TABLE 6 2,3-butanediol productivity in a batch culture at varyingspecific CO uptakes. Average specific CO Average 2,3-butanediol Dayuptake (mmol/g/min) production rate 1-3 0.8  1.5 g/L/day 5-6 0.1 0.25g/L/day

Total 2,3-butanediol accumulation over 7.5 days was approx 7.5 g/L. Itis recognised that 2,3-butanediol is produced at low levels at lowerspecific CO uptake rates. However, when the gas is supplied such thatthe CO uptake rate can be maintained over 0.4 mmol/g/min, 2,3-butanediolproductivity increase significantly. In this instance, the specific COuptake is maintained at an average of 0.8 mmol/g/min over several daysand 1,3-butanediol is produced at a rate in excess of 1 g/L.

Example 5 Batch fermentation in CSTR

Approximately 1.3 L of solution A was transferred into a 2 L fermenterand sparged with nitrogen. H₃PO₄ (85% solution, 2.025 mL, 30 mM) wasadded and the pH adjusted to 5.3 using concentrated NH₄OH(aq). SolutionB (13.5 ml) was added and the solution sparged with N2.Chromium(II)chloride was added until the ORP of the solution decreasedto approximately −150 mV. Resazurin (1.35 mL of a 2 g/L solution) wasadded. Sodium polysulfide (2.85 ml of a 6M solution) was added and thesolution sparged with N2 for 12 hours before switching to CO containinggas (1% H2; 14% N2; 70% CO; 15% CO2). The pH was adjusted to 5.5 withconcentrated HCl before the addition of metal sulfide solution 1 (150ml). The solution was sparged with the CO containing gas for a further30 minutes before inoculation with an actively growing Clostridiumautoethanogenum culture at a level of approximately 5% (v/v). Again, gasflow rate was adjusted to ensure the microbial culture was not limitedin CO in order to maintain a high specific CO uptake. Results of thefermentation are shown in Table 7.

TABLE 7 2,3-butanediol productivity in a batch culture at varyingspecific CO uptakes. Average CO uptake Average 2,3-butanediol Day(mmol/g/min) production rate 1-4 0.85  0.8 g/L/day 5-6 0.3 0.25 g/L/day

Total 2,3-butanediol after approx 6 days was 5 g/L. Once again, elevatedspecific CO uptake results in significantly higher 2,3-butanediolproductivity of at least 0.5 g/L/day.

Example 6 Batch Fermentation in CSTR

Approximately 1.3 L of solution A was transferred into a 1.5 L fermenterand sparged with nitrogen. H₃PO₄ (85% solution, 2.25 mL) was added andthe pH adjusted to 5.3 using concentrated NH₄OH(aq). Solution B (15 ml)was added and the solution sparged with N2. Chromium(II)chloride wasadded until the ORP of the solution decreased to approximately −150 mV.Resazurin (1.5 mL of a 2 g/L solution) was added. Sodium polysulfide(1.5 ml of a 3M solution) was added and the solution sparged with N2 for1 hour. 0.1M solutions of FeCl₂ (3.75 mL), CoCl₂ (1.875 mL), NiCl₂(1.875 mL), H₃BO₃ (0.375 ml), Na₂MoO₄ (0.375 ml), MnCl₂ (0.375 ml),Na₂WO₄ (0.375 ml) and ZnCl₂ (0.1875 ml) were added and the solutionsparged with CO containing gas (50% H2; 32% CO; 4% CO2; 32% CH4). The pHwas adjusted to 5.5 with concentrated HCl before the addition ofSolution C (150 ml). The solution was sparged with the CO containing gasfor a further 30 minutes before inoculation with an actively growingClostridium autoethanogenum culture at a level of approximately 5%(v/v). Gas flow rate was adjusted to ensure the microbial culture wasnot limited in CO in order to maintain a high specific CO uptake.Results of the fermentation are shown in Table 8.

TABLE 8 2,3-butanediol productivity in a batch culture at varyingspecific CO uptakes. Average CO uptake Average 2,3-butanediol Day(mmol/g/min) production rate 0-4  0.07 0 5-14 0.15 0.2 g/L/day

Total 2,3-butanediol concentration after 4 days was approximately 3 g/L.While the rates achieved are less than previous fermentations (examples4 and 5), the substrate stream comprises a substantial portion ofhydrogen. The results show that 2,3-butanediol is produced when using amixed CO/H2 substrate.

Example 7 Continuous Fermentation in Continuous Stirred Tank Reactor

Media was prepared at pH 5.5 as follows. All ingredients in Solution D,with the exception of Cysteine-HCl were mixed in 400 ml distilled water.This solution was made anaerobic by heating to boiling and allowing itto cool to room temperature under a constant flow of 95% CO, 5% CO2 gas.Once cool, the Cysteine-HCl was added and the pH of the solutionadjusted to 5.5 before making the volume up to 1000 ml; anaerobicity wasmaintained throughout the experiments.

A five-litre bioreactor was charged with 4.9 L of LM33 media prepared asdescribed above. The gas was switched to CO containing gas (1% H2; 14%N2; 70% CO; 15% CO2) at atmospheric pressure prior to inoculation with100 ml of a Clostridium autoethanogenum culture. The bioreactor wasmaintained at 37° C. stirred at 200 rpm at the start of the culture.During the growth phase, the agitation was increased to 400 rpm. The pHwas adjusted to 5.5 and maintained by automatic addition of 5 M NaOH.Fresh anaerobic media was continuously added into the bioreactor tomaintain a defined biomass and acetate level in the bioreactor. 2,3butanediol productivity is highlighted in Table 9.

TABLE 9 2,3-butanediol productivity in a continuous culture. Average COuptake Average 2,3-butanediol Day (mmol/g/min) production rate  1-87 0.3<0.1 g/L/day 90-92 0.6 1.2 g/L/day 93-95 0.4 0.87 g/L/day

During the first 89 days of continuous operation, the fermenter wasoperated under CO limited conditions and minimal 2,3-butanediol wasproduced. However, around day 88, gas flow was increased, such thatspecific CO uptake increased. At this stage, 2,3-butanediol productivityincreased significantly to at least 1.2 g/L/day. Around day 92, the gasflow was reduced such that the specific uptake of the culture decreasedto around 0.4 mmol/g/min and the 2,3-butanediol productivity alsodropped. However, even at an average specific uptake of approximately0.4 mmol/g/min, 2,3-butanediol productivity remained at least 0.5g/L/day.

Example 8 Batch Fermentation in CSTR

Approximately 1.3 L of solution A was transferred into a 2 L fermenterand sparged with nitrogen. H₃PO₄ (85% solution, 1.5 ml) was added andthe pH adjusted to 5.3 using conc.

NH₄OH(aq). Solution B (15 ml) was added and the solution sparged withN2. Chromium(II)chloride was added until the ORP of the solutiondecreased to approximately −150 mV. Sodium polysulfide (1 ml of a 3Msolution) was added and the solution sparged with N2 for 12 hours. 0.1Msolutions of FeCl₂ (3.75 mL), CoCl₂ (1.875 mL), NiCl₂ (1.875 mL), H₃BO₃(0.375 ml), Na₂MoO (0.375 ml), MnCl₂ (0.375 ml), Na₂WO₄ (0.375 ml) andZnCl₂ (0.2 ml) were added and the solution sparged with CO containinggas (1% H2; 14% N2; 70% CO; 15% CO2). Resazurin (1 mL of a 2 g/Lsolution) was added. The pH was adjusted to 5.5 with concentrated HCland the solution was sparged with the CO containing gas for a further 30minutes before inoculation with an actively growing Clostridiumautoethanogenum culture at a level of approximately 5% (v/v). Table 10shows the accumulated 2,3-butanediol product in a fermenter afterapproximately 2 weeks operation. The specific CO uptake rates have beencorrected for the culture viability. Culture viability was determinedusing the methods described in WO2009/022925, which is incorporatedherein by reference.

TABLE 10 2,3-butanediol accumulation after 14 days batch fermentation.2,3-butanediol Specific CO uptake (accumulated Day (mmol/g/min) product)13 0.6 8.67 g/L 14 0.5 9.27 g/L

Over the 24 hour period from day 13-14, the specific CO uptake wasmaintained at approximately 0.5 mmol/g/min and 2,3-butanediolproductivity was 0.6 g/L/day.

Example 9 Gene Regulation of 2,3-Butanediol Production in LZ1560

Samples were taken from three fermentations to determine gene expressionduring 2,3-butanediol production. One sample was taken from the batchfermentation described in Example 8 on day 13, wherein productsincluding ethanol and 2,3-butanediol were being produced. The sample isdesignated R12 in the results hereinafter. The second sample was takenfrom a batch fermentation producing both ethanol and 2,3-butanediol. Thesample is designated R11 in the results. The third sample (R2) was takenfrom the continuous fermentation operating under similar conditions asExample 7 on days 1-89. The microbial culture was CO limited and thefermentation broth had a stable acetate concentration of approximately13 g/L, ethanol concentration of less than 1 g/L and insignificantamounts of 2,3-butanediol. Real-Time PCR was used to determine whethergenes were upregulated or downregulated relative to R2.

RNA Extraction and cDNA Synthesis Procedure:

Total RNA was isolated from approximately 2.5×10⁹ bacterial cells usingAurum Total RNA Fatty and Fibrous Tissue Kit (Biorad). On-column DNasewas digested using the RNase-free DNase set (Biorad). Total RNA wasquantified using spectrophotometer and its purity (measured by theA_(260/280) ratio) was determined before cDNA synthesis using iScriptSelect cDNA synthesis kit (Biorad).

Real-Time PCR Procedure:

Primers for Real-Time PCR, were designed using the freeware Primer3based on LanzaTech's proprietary in-house genome sequence. Real-Time PCRreaction mixtures containing 12.5 μL 2×SYBR Green PCR Master Mix(Biorad), 1.54 of each of 1 μM primer forward and reverse, 5 μL of 10×diluted cDNA template, and sterile water to a total volume of 25 μL wereassembled. The mixtures were heated to 95° C. for 3 minutes, followed by40 cycles of 95° C. for 15 seconds, 55° C. for 15 seconds and 72° C. for30 seconds. For detection of primer dimerisation or other artifacts ofamplification, a melting-curve analysis was performed immediately aftercompletion of the real-time PCR (38 cycles of 58° C. to 95° C. at 1°C./seconds). All reactions were performed in triplicate. Quantificationof gene expression was performed using a MyiQ Single Colour Real-TimePCR Detection System (Biorad) and real-time data were analyzed using theiQ5 optical system software (Biorad).

Results:

The raw Ct values, together with the relative gene expression andstandard errors, generated from the Real-Time PCR assay are presented inTable 11. RNA polymerase beta chain (rpoB) was selected as referencegene for normalizing gene expression. Relative quantification using theComparative ΔC_(T) method was used to calculate the relative geneexpression of 2,3BDH. The acetate-producing culture (R2) was selected ascalibrator (reference standard) in all analysis.

TABLE 11 Derivation of relative gene expression values from raw Ct data.Relative expressions were normalized by rpoB and calibrated usingReactor 2. SE = standard error of the mean. (Any relative expressionabove 1 shows up regulation) Relative Relative expression Genes ReactorRaw Ct Mean Ct SD Ct expression SE rpoB 11 36.36 35.4 0.846 NA NA 1135.07 11 34.77 12 32.47 32.7 0.258 NA NA 12 32.65 12 32.98 2 31.76 31.760.051 NA NA 2 31.81 2 31.71 2,3BDH 11 27.01 26.57 0.422 4.75 1.8 1126.53 11 26.17 12 23.23 23.18 0.038 7.64 0.8 12 23.15 12 23.17 2 24.8125.18 0.559 1 0.23 2 24.91 2 25.82

Real-time PCR data presented in this study shows that 2,3-butanediolgene expression is significantly higher in solventogenic cultures(R11/R12) compared to acetogenic cultures (R2). The microbial culture ofR12, which was producing approximately 0.6 g/L/day 2,3-butanediol at thetime of cell harvest, shows the highest gene up-regulation (7.64±0.8fold), relative to R2. This is followed by R11 with 4.75±1.8 foldup-regulation of the gene, which had a total 2,3-butanediol productionof 1.53 g/L, when the cells were harvested.

FIG. 1 shows relative gene expression of 2,3-butanediol dehydrogenase(2,3BDH) in three fermenters (R11, R12 and R2). Acetate-producing R2 isselected as calibrator and gene expression was normalized using rpoB asreference gene. Error bar=standard error of mean. N=3. Clearly,2,3-butanediol dehydrogenase is upregulated in microbial cultures thatproduce 2,3-butanediol. The microbial culture in R2 has a specific COuptake of approximately 0.3 mmol/g/min, whereas the culture in R12 has aspecific uptake of approximately 0.6 mmol/g/min. Increasing the amountof CO provided to the culture results in an increase in CO uptake and asubsequent increase in 2,3-butanediol dehydrogenase gene expression. Theincrease in 2,3-butanediol dehydrogenase gene expression results in anincrease in overall 2,3-butanediol productivity.

The invention has been described herein with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. Those skilled in the art willappreciate that the invention is susceptible to variations andmodifications other than those specifically described. It is to beunderstood that the invention includes all such variations andmodifications. Furthermore, titles, headings, or the like are providedto enhance the reader's comprehension of this document, and should notbe read as limiting the scope of the present invention.

The entire disclosures of all applications, patents and publications,cited above and below, if any, are hereby incorporated by reference.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in the UnitedStates of America or any country in the world.

Throughout this specification and any claims which follow, unless thecontext requires otherwise, the words “comprise”, “comprising” and thelike, are to be construed in an inclusive sense as opposed to anexclusive sense, that is to say, in the sense of “including, but notlimited to”.

1. A method of producing butanediol by microbial fermentation, themethod including: a. providing a substrate; b. in a bioreactorcontaining a culture of one or more micro-organisms, anaerobicallyfermenting the substrate to produce butanediol.
 2. A method according toclaim 1, wherein the substrate comprises CO.
 3. A method according toclaim 2, wherein the butanediol is 2,3-butanediol.
 4. A method accordingto claim 3, wherein the substrate comprising CO is provided at asufficient level, such that 2,3-butanediol is produced.
 5. A methodaccording to claim 3, wherein the substrate comprising CO is providedsuch that a specific rate of CO uptake of at least 0.4 mmol CO/gram drycells weight of bacteria/minute by the culture is maintained.
 6. Amethod according to claim 5, wherein the substrate is provided such thata specific uptake rate of at least 0.6 mmol CO/g/min is maintained.
 7. Amethod according to claim 5, wherein the substrate is provided such thata specific uptake rate of at least 0.8 mmol CO/g/min is maintained.
 8. Amethod according to claim 5, wherein the substrate is provided such thata specific uptake rate of at least 1.0 mmol CO/g/min is maintained.
 9. Amethod of producing 2,3-butanediol by microbial fermentation, the methodincluding: a. providing a substrate; b. in a bioreactor containing aculture of one or more micro-organisms, anaerobically fermenting thesubstrate, wherein one or more micro-organisms includes one or more2,3-butanediol dehydrogenase genes; c. upregulating the 2,3-butanedioldehydrogenase gene(s), such that 2,3-butanediol is produced by themicro-organism(s).
 10. A method according to claim 9, wherein thesubstrate comprises CO.
 11. A method according to claim 10, wherein thesubstrate comprising CO is provided at a sufficient level, such that2,3-butanediol is produced.
 12. A method according to claim 10, whereinthe substrate comprising CO is provided such that a specific rate of COuptake of at least 0.4 mmol CO/gram dry cells weight of bacteria/minuteby the culture is maintained.
 13. A method according to claim 9, whereinone or more micro-organism(s) is Clostridium autoethanogenum.
 14. Amethod according to claim 2, wherein the substrate comprising CO isgaseous and comprises at least about 15% to about 100% CO by volume. 15.A method according to claim 2, wherein the substrate comprising COcomprises gas obtained as by-product from an industrial process.
 16. Amethod according to claim 15, wherein the substrate comprising COcomprises gas obtained from a steel mill.
 17. A method according toclaim 1 wherein the butanediol is 2,3-butanediol and the cultureincludes a culture of Clostridium autoethanogenum.
 18. A methodaccording to claim 17, wherein the substrate comprises one or morecarbohydrates.
 19. A method providing a substrate according to claim 18,wherein the method includes an additional step of providing a substratecomprising CO before, and/or at the same time, and/or after the step ofproviding a substrate comprising one or more carbohydrates. 20.2,3-Butanediol produced by the method of claim
 3. 21-23. (canceled)